Echinocandin process

ABSTRACT

This invention relates to an improved process for the minimization of acid-catalyzed reactions of certain echinocandins of the kind disclosed in U.S. Pat. No. 5,378,804. The process involves the use of a boronic acid.

This applicatian is related to PCT application PCT/US02/12756, filedApr. 8, 2002.

BACKGROUND OF THE INVENTION

This invention relates to an improved process for the minimization ofacid-catalyzed reactions of certain echinocandins of the kind disclosedin U.S. Pat. No. 5,378,804. The echinocandin compounds disclosed in thepatent have been prepared as described in the patent and in patentsclaiming improvements of the process. U.S. Pat. No. 5,552,521 disclosesa three-step process for preparing the compounds of the invention. U.S.Pat. No. 5,936,062 discloses an improvement of the three-step processusing a boronate intermediate. Articles in the Journal of OrganicChemistry, 1999, 64, 2411–2417 and J. Med. Chem. 1994, 37, 222–225,describe an amide to nitrile dehydration of similar echinocandins usingcyanuric chloride. However, the previous processes resulted in theformation of unwanted benzylic substituted derivatives of the desiredcompound. The instant invention results in increased yield of thedesired product while minimizing the acid-catalyzed reaction at thebenzylic center as well as acid-catalyzed epimerization at the benzyliccenter. Additionally, the process allows for the use of moderate tostrong acid catalysts previously determined to be ineffective atcatalyzing the reaction in a highly chemo-selective manner. Theimprovement permits the use of considerably less acid to complete theformation of the desired compound. Additionally, the α/βstereoselectivity for the phenylsulfide formation reaction is greatlyincreased.

SUMMARY OF THE INVENTION

This invention is directed to a process for the minimization of certainimpurities generated by acid-catalyzed reactions of certainechinocandins. In particular, the invention describes an improvement tothe process of preparing certain sulfide-substituted echinocandinsformed as intermediates in the preparation of the compound of thestructure

This compound has been found useful in treating life-threatening fungalinfections.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a process for minimizing of certain impuritiesgenerated by the acid-catalyzed reactions of certain echinocandins,which results in an improvement to the process for the preparation ofcertain sulfide-substituted echinocandins required for the preparationof the compound of the structure

or its pharmaceutically acceptable salt, hydrate or solvate thereof.

Additionally, the invention relates to an improvement in the preparationof the compound of the structure 1

or its pharmaceutically acceptable salt, hydrate or solvate thereof,from the starting material used to prepare the compound of structure I.

A process for the preparation of a Compound 1 of structure

or its pharmaceutically acceptable salt, hydrate or solvate thereof,which comprises the steps of:

-   a) reacting an amide Compound 2 of the structure

-    with a boronic acid or borate in a solvent to obtain a boronate or    borate, respectively; and-   b) dehydrating the boronate or borate with cyanuric chloride in a    second solvent to produce a nitrile Compound 1

-    with concomitant formation of a minimal amount of an epimer of the    nitrile Compound 1, Compound 3 having the structure

Prior processes resulted in the formation of about 3–5% of Compound 3,the benzylic hydroxyl epimer of the nitrile Compound 1. The instantinvention reduces the acid-catalyzed epimerization of the benzylichydroxyl group to <1%.

Compound 2 can be produced by cultivating Glarea lozoyensis (formerlyidentified as Zalerion arboricola) ATCC 20868 in a nutrient mediumenriched in mannitol as the primary source of carbon, as described inU.S. Pat. No. 5,021,341, issued Jun. 4, 1991. Compound 2 is claimed inU.S. Pat. No.5,202,309 that issued Apr. 13, 1993. Compound 2 can also beproduced by cultivating Zalerion arboricola in a nutrient mediumenriched the unnatural amino acid selected from the group consisting of:3,4-dehydroproline, L-azetidine-2-carboxylic acid, L-proline or mixture,as described in U.S. Pat. No. 5,194,377 issued on Mar. 16, 1993.

Step a involves reaction of Compound 2 and a boronic acid in a firstsolvent. About 100 to about 200 mole % of the boronic acid can beemployed, and about 200 mole % of the boronic acid is preferred. Anyboronic acid or borate can be used to produce the desired result.Examples of such boronic acids or borates are: phenylboronic acid,3-nitrophenylboronic acid, 4-methoxyphenylboronic acid,3,5-bis(trifluoromethyl)phenylboronic acid, n-butylboronic acid,trimethyl borate and triethylborate. A preferred boronic acid is phenylboronic acid. Examples of such first solvent are tetrahydrofuran, CH3CNor a mixture therefrom.

When formation of the borate/boronic is complete in Step a, the water isremoved from the reaction mixture. The water is removed by azeotropicdistillation of the reaction solvent with concomitant addition of drysolvent or by passing the refluxate through a bed of molecular sievesuntil a ratio of less than about 20 mole % water to Compound 2 isobtained.

After removal of the water from the reaction mixture, the solvent isremoved in vacuo and subsequent dissolution of the borate/boronatederivative with a second solvent. Suitable second solvent(s) such as dryN,N-dimethylformamide (DMF), tetrahydrofuran, or N-methylpyrrolidinone(NMP) or mixtures thereof can be used. The preferred solvent is amixture of N-methylpyrrolidinone and N,N-dimethylformamide.

Step b involves dehydration of the borate/boronate derivative in thesutiable second solvent with cyanuric chloride or other appropriatereagent to yield the nitrile. The amount of cyanuric chloride used iscrucial to limit the formation of the undesired hydroxyl epimer at thebenzylic position of the homo-tyrosine portion of the cyclic peptide.About 100 to about 300 mole % of cyanuric chloride can be employed, andabout 180 to about 230 mole % is the preferred range.

A preferred embodiment of this invention is the process wherein about200 mole % phenylboronic acid in tetrahydrofuran is used in Step a atambient temperature, followed by removal of water by azeotropicdistillation of the refluxate through molecular sieves (3 Å). Thereaction mixture is then dried and the solvent removed in vacuo. Theresulting solids were dissolved in the second solvent(s) a 9:1N-methylpyrrolidinone/N,N-dimethylformamide volume to volume mixture anddehydrated with 230 mole % of cyanuric chloride. The cyanuric chloridewas added at −13° C. and the reaction mixture was aged at −13° C. to−23° C. for 20 hours. These conditions resulted in a chemical yield ofabout 84% with no benzylic hydroxyl epimer (Compound 3) detected by HPLCanalysis.

In a second embodiment of this invention, there is disclosed an improvedprocess for minimizing the acid-catalyzed reaction of R³—SH at thebenzylic center which results in a significantly lower formation of theundesired benzylic-substituted bissulfide derivative, 5B. Priorprocesses resulted in >9% of the undesired benzylic-substitutedbis(phenylsulfide) derivatives, 5B (wherein R³—SH is thiophenol). Thisprocess, wherein R³—SH is thiophenol, also results in an increase inchemical yield to about 92% to about 95% with the formation of onlyabout 2–3% of the undesired benzylic-substituted bis(phenylsulfide)derivatives. The improved process allows for the use of strong acidcatalysts previously determined to be ineffective due to excessivesubstitution or epimerization of the benzylic hydroxyl group. Inaddition, the improved process allows for the use of significantly lessacid to complete the sulfide formation. The sulfide compound, 5A is akey intermediate in the preparation of the compound of Structure I.

This embodiment of the invention discloses, a process for preparingCompound 5A of the structure

or pharmaceutically acceptable salt, hydrate or solvate thereof, whileminimizing the formation of Compounds 5B and Compound 5C of thestructure

wherein

-   -   R is C(═O)NH₂, CH₂NH₃+X— or CN;    -   X— is Cl⁻, CF₃COO⁻, CH₃COO⁻, CF₃SO₃ ⁻, HSO₄ ⁻; and    -   R₃ is aryl, wherein aryl is defined as phenyl or naphthyl,        substituted or unsubstituted with C₁–C₆-alkyl, C₁–C₆-alkoxy,        halo (Br, Cl, F, I), (CH₂)aryl, heteroaryl, wherein heteroaryl        is defined as 5-membered ring, 6-membered ring, 5,6-fused ring,        or 6,6-fused ring bearing 1 to 3 heteroatoms selected from N, O,        or S optionally substituted with C₁–C₆-alkyl, or C₁–C₆-alkoxy;        which comprises the steps of:    -   A) reacting Compound 4 of the structure

-   -    with a boronic acid or borate in a solvent to afford        boronate(s) or borate(s), respectively, of compound 4; and    -   B) reacting the boronate or borate with a thiol, R₃SH, wherein        R₃ is as defined above, and an acid to afford Compound 5A with        minimal amounts of Compounds 5B and 5C.

The sulfide, Compound 5A is prepared by formation of the boronate/boratein Step A which comprises reaction of Compound 4 (Compound 2,R═C(═O)NH₂; Compound 1, R═CN or Compound 6, R═CH₂NH₃+X—) with a boronicacid, R₁B(OH)₂, or borate, (R₂O)₃B, in a solvent; followed by reactionof the boronate/borate produced in Step A with a thiol, R₃SH, and anacid, R₄—H+, in Step B to produce the sulfide, Compound 5A.

Solvent(s) such as tetrahydrofuran, acetonitrile, or mixtures thereofcan be used in reaction Step A. Any boronic acid or borate is expectedto produce the desired results. Examples of such boronic acid or borateinclude, but are not limited to, phenylboronic acid,3-nitrophenylboronic acid, 3,5-bis(trifluoromethyl)phenyl-boronic acid,4-methoxyphenylboronic acid, n-butylboronic acid, trimethyl borate andtriethyl borate. The reaction is carried out at a temperature range ofabout 20° C. to about 66° C. and for a reaction time of about 15 minutesto about 12 hours.

The Compound 4, wherein R represents CH₂NH₃+X— (also referred to asCompound 6) is prepared by chemical or catalytic reduction of thenitrile compound, Compound 1. Chemical reduction can be carried outusing sodium borohydride, aluminum hydride, diborane, diisobutylaluminum hydride and the like. Catalytic reduction may also be employedusing hydrogenation with a variety of catalysts including palladium oncarbon, platinum oxide, or rodium on alumina. See U.S. Pat. No.5,939,384 and the Journal of Organic Chemistry, 1999, 64, 2411–2417.

A preferred embodiment of this invention is the process Step A asrecited above, wherein the boronic acid is phenylboronic acid and thesolvent is CH₃CN.

Following the formation of the boronate/borate in Step A, theboronate/borate product is combined with a thiol and suitable acid inStep B to yield the desired sulfide, Compound 5A in a highlystereoselective and chemoselective manner. Any thiol is expected toproduce the sulfide intermediate. R₄—H+ represents a suitable acid, amoderate to strong acid that would be expected to produce the sulfideintermediate in good yield. Examples of such moderate to strong acidsinclude, but are not limited to, trifluoroacetic acid, methanesulfonicacid, p-toluenesulfonic acid, and trifluoromethanesulfonic acid.

A preferred embodiment of this invention is the process Step A asrecited above, wherein the thiol is thiophenol and the R₄—H+ istrifluoromethanesulfonic acid.

When reaction Step B is complete, the mixture is quenched with water ordilute base and, following an annealing step, the sulfide intermediatesare isolated by filtration.

The amount of boronic acid or borate used is crucial to the rate ofdisplacement, as well as to the formation of the undesirablebis(sulfide) derivative (Compound 5B) and benzylic hydroxyl epimer(Compound 5C). It was found that from about 100 to about 900 mole % of aboronic acid or a borate gave the best yield of Compound 5A with about100 to about 300 mole % of the boronic acid being the preferred range.

The amount of thiol used in the sulfide formation step is also criticalto the yield of Compound 5A. About 200 to about 500 mole % of thiophenolprovided the best yield of the phenylsulfide intermediate (Compound 5A).

The amount of R₄—H+ used is also critical to the yield of Compound 5Aand formation of the undesired Compounds 5B and 5C. It was found thatabout 200 to about 4600 mole % of a moderate to strong acid catalystgave the best yield. Additionally, the conditions provide optimalprocess aging time, chemoselectivity, stereoselectivity, and the bestmeans of producing an easily filtered product.

A preferred embodiment of the invention is the process as recited abovewherein Compound 4 is reacted with about 200 mole % of phenylboronicacid in acetonitrile at ambient temperature for about 0.5 hours followedby reaction of the boronate with 300 mole % thiophenol and about 300mole % trifluoromethanesulfonic acid at about −13° C. HPLC analysisshowed a chemical yield of about 95% with concomitant formation of about3 area % of the bis(phenylsulfide) and 0.1 area % of the benzylichydroxyl epimer, Compounds 5B and 5C respectively. The reaction mixtureis diluted to 90% acetonitrile/10% water (v/v) with water containing 300mole % sodium acetate. The product was isolated by filtration after anannealing step. The phenylsulfide intermediate was isolated in about91%. yield and contained 1.1 area % of the undesiredbenzylic-substituted bis(phenylsulfide) derivative (Compound 5B) and<0.1 area % of the undesired benzylic hydroxyl epimer (Compound 5C) asanalyzed by HPLC.

An additional embodiment of the process for preparing the sulfideCompound 5A, is the process which includes the additional steps of:

-   -   (i) quenching the reaction mixture containing Compound 5A with        aqueous base and cooling the reaction mixture to precipitate        Compound 5A as a suspension of fine amorphous particles of        Compound 5A;    -   (ii) warming the suspension of Compound 5A to about 15 to about        25° C. over about 1.5 to 2.0 hours to produce larger amorphous        particles of Compound 5A and aging the suspension of Compound 5A        for about 20 to about 30 minutes at about 15 to about 25° C.;    -   (iii) cooling the suspension with larger amorphous particles of        Compound 5A to 0° C. and aging the suspension of Compound 5A for        about 1 hour; and    -   (iv) filtering the suspension of larger amorphous particles of        Compound 5A to isolate larger amorphous particles of Compound        5A.

The invention is described in greater detail in the following examplesin which all parts, preparations, ratios and percentages are by weightunless otherwise indicated.

EXAMPLE 1

Synthesis of Compound 1

The amide, Compound 2, (1.0 g, 0.94 mmole) and phenylboronic acid (235mg, 1.90 mmole) were added to dry tetrahydrofuran (10 mL) and thesuspension was agitated until all solids dissolved (40 min). Theresulting solution was dried to <20 mol % water: Compound 2 by passingthe refluxate through a bed of molecular sieves (3A). A portion of thedried solution (1.0 mL) containing Compound 2 boronate (120 mg, 0.12mmole) was transferred to a 25 mL flask where the tetrahydrofuran wasremoved under vacuum. The resulting solid was dissolved in 4.0 mL dry1-methyl-2-pyrrolidinone and 0.45 mL dry N,N-dimethylformamide atambient temperature. Cyanuric chloride (50 mg, 0.27 mmole) was thenadded at −13° C. with stirring. The reaction was aged for 2.5 hours at−13° C. followed by 18 hours at −23° C. HPLC analysis (210 nm) showed 3area % Compound 2 and an 84% yield of Compound 1. Compound 3 was notdetected by HPLC at this time nor was it observed after an additional 26hours of the reaction at −20° C.

EXAMPLE 2

Synthesis and Isolation of Compound 5A (R═C(═O)NH₂, R₃=phenol)

Compound 4 (R═C(═O)NH₂, 23.1 g, 21.7 mmole) and phenylboronic acid (5.72g, 46.9 mmole) were added to dry acetonitrile (750 mL). The suspensionwas stirred at ambient temperature for 0.5 hours and then cooled to −6°C. where thiophenol (7.24 mL, 70.4 mmole) was added.Trifluoromethanesulfonic acid (6.23 mL, 70.4 mmole) was then added over10 minutes while maintaining a temperature of −13° C. The reactionprogress was monitored by HPLC until the ratio of startingmaterial/product was 1:99 (2 hours). The chemical yield of phenylsulfideintermediate for the reaction was 95% as a 60:1 mixture of α- andβ-diastereomers by HPLC assay. The undesired benzylic-substitutedbis(phenylsulfide) derivative (Compound 5B, R═C(═O)NH₂, R₃=phenyl) andthe undesired benzylic hydroxyl epimer (Compound 5C, R═C(═O)NH₂,R₃=phenyl) were present at 3.2 HPLC area % (210 nm) and 0.1 HPLC area %(210 nm), respectively. At 2.5 hours, a solution of NaOAc₃H₂O (9.58 g,70.4 mmole) in 80 mL water was added at a rate so as to maintain thetemperature below −9° C. The product precipitated as 1–2 μm amorphousparticles. The suspension was warmed to 19° C. over 1.25 hours duringwhich the amorphous precipitate turned over to 5–25 μm amorphousparticles. The suspension was then aged 20 minutes at 19° C. and thencooled to 0° C. and aged 1 hour. The solids were removed by filtrationand washed with 3×100 mL of 9:1 acetonitrile:water (v/v). The materialwas then dried under a nitrogen flow. The assay yield of thephenylsulfide was 22.7 g (91%) as a 180:1 mixture of α- andβ-diastereomers. The solid contained 1.1 HPLC area % (210 nm) of theundesired benzylic-substituted bis(phenylsulfide) derivative (Compound5B, R═C(═O)NH₂, R₃=phenyl) and <0.1 HPLC area % (210 nm) of theundesired benzylic hydroxyl epimer (Compound 5C, R═C(═O)NH₂, R₃=phenyl)by BPLC analysis.

1. A process for preparing Compound 5A of the structure

while minimizing the formation of Compounds 5B and Compound 5C of thestructure

or their pharmaceutically acceptable salts, hydrates or solvatesthereof, which comprises the steps of: A) reacting Compound 4 of thestructure

 wherein R is C(═O)NH₂, CH₂NH₃+X— or CN; X—is Cl⁻, CF₃COO⁻, CH₃COO⁻,CF₃SO₃ ⁻, HSO₄ ⁻; and R₃ is aryl, wherein aryl is defined as phenyl ornaphthyl, substituted or unsubstituted with C₁–C₆-alkyl, C₁–C₆-alkoxy,halo (Br, Cl, F, I), (CH₂)aryl, heteroaryl, wherein heteroaryl isdefined as 5-membered ring, 6-membered ring, 5,6-fused ring, or6,6-fused ring bearing 1 to 3 heteroatoms selected from N, O, or Soptionally substituted with C₁–C₆-alkyl, or C₁–C₆-alkoxy; with a boronicacid or borate in a solvent to afford boronate(s) or borate(s),respectively, of compound 4; and B) reacting the boronate or borate witha thiol, R₃SH, wherein R₃ is as defined above, and an acid in the amountof 1 eq. of the thiol R₃SH to afford Compound 5A with minimal amounts ofCompounds 5B and 5C.
 2. The process of claim 1, further comprising theadditional steps of: (i) quenching the reaction mixture containingCompound 5A with aqueous base and cooling the reaction mixture toprecipitate Compound 5A as a suspension of fine amorphous particles ofCompound 5A; (ii) warming the suspension of compound 5A to about 15 toabout 25° C. over about 1.5 to 2.0 hours to produce larger amorphousparticles of Compound 5A and aging the suspension of Compound 5A forabout 20 to about 30 minutes at about 15 about 25° C.; (iii) cooling thesuspension with larger amoiphous particles of Compound 5A to 0° C. andaging the suspension of Compound 5A for about 1 hour; and (iv) filteringthe suspension of larger amorphous particles of Compound 5A to isolatelarger amorphous particles of Compound 5A.
 3. The process of claim 1,wherein the boronic acid or borate in Step A is selected from the groupconsisting of: myl boronic acid, wherein aryl is defined as phenyl, 1-or 2-naphthyl, 9-anthryl or phenmntbryl, unsubstituted or substitutedwith 1, 2 or 3 substitutents selected from the group consisting of:nitro, C₁–C₆-alkyl, C₁–C₆-alkoxy, halo (Br, Cl, F, I), andtrifluoromithyl, C₁–C₆-alkyl boronic acid, or tri(C₁–C₆-alkyl) borate.4. The process of claim 3, wherein the solvent used in Step A istetrahydrofuran, CH₃CN or a mixture therefrom.
 5. The process of claim 4wherein the thiol, R₃SH used in Step B is selected from the groupconsisting of: thiophenol, 4-methoxythiophenol, benzylthiol,1-methylixnidazol-2-ylthiol, and benziniidazol-2-ylthiol.
 6. The processof claim 5, wherein the acid used in Step B is selected from the groupconsisting of: trifluoroacetic acid, methanesulfonic acid,p-tohzenesnlfonic acid, and trifluoromethaneaulfonic acid.
 7. Theprocess of claim 6, wherein the boronic acid used in Step A isphenylboronic acid.
 8. The process of claim 7, wherein the solvent usedin Step A is CH₃CN.
 9. The process of claim 8, wherein the acid used inStep B is trifluoromethanosulfonic acid.
 10. The process of claim 9,wherein the thiol, R₃SH used in Step B is thiophenol.
 11. The process ofclaim 10, wherein about 100 to about 300 mole % of phenylboronic acid isused in Step B.
 12. The process of claim 11, wherein about 200 to about500 mole % of the thiophenol is used in Step B.
 13. The process of claim12, wherein the reaction in Step B takes place at a temperature of about−5° C. to about −20° C.
 14. The process of claim 8, wherein the acidused in Step B is trifluoroacetic acid.
 15. The process of claim 14,wherein the thiol, R₃SH used in Step B is thiophenol.
 16. The process ofclaim 15, wherein about 300 mole % of phenylboronic acid is used in StepB.
 17. The process of claim 16, wherein about 200 to 500 mole % of thethiophenol is used in Step B.
 18. The process of claim 17, wherein thereaction in Step B takes place at a temperature of about −10° C. to +5°C.